1. Field of the Invention
The present invention relates to DNA fragments that confer resistance to porphyrin-accumulating type herbicides on plant and algal cells, plasmids and microorganisms that contain these DNA fragments, methods for conferring resistance to porphyrin-accumulating type herbicides onto plant and algal cells by using these DNA fragments, and plants and algae into which these DNA fragments have been introduced for the purpose of conferring resistance to such herbicides thereon.
2. Description of Related Art
A group of widely-known compounds used as active ingredients of some varieties of commercially- and otherwise-available herbicides exhibit herbicidal activity in the presence of light, but exhibit no herbicidal activity in darkness. This has led to their common designation as light-dependent or porphyric herbicides. It has recently been shown that these herbicides induce high levels of porphyrin accumulation in plants and algae, and thus they are now designated as xe2x80x9cporphyrin-accumulating type herbicidesxe2x80x9d [Zoku, Iyakuhin-no-Kaihatsu, (translation: xe2x80x9cThe Development of Medical Drug Products; continuationxe2x80x9d) vol. 18; Development of Agricultural Chemicals II, chapter 16, section 16-1, Hajime Iwamura, Tamio Ueno and Katsuzo Kamoshita, eds., Hirokawa Shoten, Tokyo, pubs.) or simply xe2x80x9cporphyric herbicidesxe2x80x9d. It was reported by Matringe, M., Camadro, J. M., Labbe, P. and Scalla, R. (Biochem J. 260:231 (1989)) and by Matringe, M., Camadro, J. M., Labbe, P. and Scalla, R. (FEBS Lett. 245:35 (1989)) that porphyrin-accumulating type herbicides (referred to below also as porphyric herbicides) inhibit isolated protopor-phyrinogen oxidase (referred to below as xe2x80x9cprotoxxe2x80x9d).
Since most crop plants do not exhibit resistance to these porphyric herbicides, it is not possible to use these herbicides on farmland when such crops are under cultivation. If it were possible to develop crops resistant to porphyric herbicides, such herbicides could be used on these crops. This would make crop management easier, and increase the value of these herbicides in, agricultural applications. For this reason, it is desirable to develop a method for conferring resistance to porphyrin-accumulating type herbicides upon crop plants.
With this goal in mind, the present inventors have investigated a mutant strain, designated RS-3, of the unicellular green alga Chlamydomonas reinhardtii which displays specific resistance to porphyric herbicides. Wild-type strains of this alga are normally highly sensitive to porphyric herbicides. The present inventors have discovered that inhibition by porphyric herbicides of protox activity in chloroplast fragments isolated from the RS-3 strain of Chlamydomonas reinhardtii was significantly lower than in chloroplast fragments from the wild-type strain. The inventors therefore constructed a genomic DNA library from total nuclear DNA isolated from the RS-3 mutant strain, and succeeded in isolating clones that contain a gene responsible for resistance to porphyric herbicides. Thus, the inventors were able to obtain DNA fragments that can confer resistance to porphyrin-accumulating type herbicides onto plant and algal cells.
Accordingly, it is an object of the present invention to provide an isolated, purified DNA fragment that confers resistance to porphyrin-accumulating type herbicides when expressed in plant or algal cells, plasmids and microorganisms containing said DNA fragment. A DNA fragment according to the present invention preferably has a nucleotide sequence of one or more portions of DNA comprising the genome of an alga, or has a nucleotide sequence highly homologous to the nucleotide sequence of DNA comprising one or more portions of the genome of an alga.
Additional objects of the present invention are a method for conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragment into said plant or algal cells, wherein said DNA fragment is expressed; and plants or algae into which said DNA fragment has been introduced, wherein said DNA fragment is expressed, thereby conferring herbicide resistance upon said plants or algae.
Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, and XhoI, and having a restriction site map as shown in FIG. 1(a);
c) having a molecular size of approximately 3.4 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI and HindIII, and having a restriction site map as shown in FIG. 1(b);
c) having a molecular size of approximately 9.9 kB; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, and KpnI, and having a restriction site map as shown in FIG. 1(c);
c) having a molecular size of approximately 10.0 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics:
a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides;
b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, BamHI, SalI, HindIII, and KpnI, and having a restriction site map as shown in FIG. 1(d);
c) having a molecular size of approximately 13.8 kb; and
d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof.
Further objects of the present invention are to provide plasmids and microorganisms containing any of the foregoing DNA fragments or biologically functional equivalents thereof, a method of conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragments or biologically functional equivalents thereof into plant or algal cells in a functionally operable manner so that said DNA fragments or biologically functional equivalents thereof are expressed in said plant or algal cells, and the expression of the DNA fragment confers resistance to porphyrin-accumulating type herbicides upon the transformed plant or algal cells. It is preferred that cells cultured in vitro that have been transformed by the DNA fragments of the invention in a functionally operable manner are resistant to a porphyrin-accumulating type herbicide at a concentration of at least 0.01 xcexcM, preferably at a concentration of at least 0.03 xcexcM, most preferably at a concentration of at least 0.1 xcexcM herbicide. When compound A or compound B are used as the test compounds, the range of concentration is preferably 0.01 to 0.3 xcexcM, more preferably 0.03 to 0.6 xcexcM, most preferably 0.1 to 0.3 xcexcM. Otherwise the range is between 0.01 to 30 xcexcM, more preferably 0.03 to 10 xcexcM, most preferably 0.1 to 3 xcexcM. The concentration of herbicide used to test resistance of transformed plants or tissues therefrom is to the high end of these ranges or even higher, and can be determined by the ordinarily skilled artisan by experimentation typical in the art. The herbicide used for testing herbicide resistance of cells in vitro or of whole transformed plants or algae is preferably a N-phenyl-tetrahydrophthalimide compound. N-(4-chloro-2-fluoro-5-propargyloxy) phenyl-3,4,5,6-tetrahydrophthalimide (compound A) or 7-fluoro-6-[(3,4,5,6,)-tetrahydrophthalimido)]-4-(2-propynyl)-1, 4-benzoxazin-3(2H)-one (referred to below as xe2x80x9ccompound Bxe2x80x9d) are especially preferred for this purpose.
Another object of the present invention is to provide plants or algae into which have been introduced in a functionally operable manner said DNA fragments or biologically functional equivalents thereof.
A still further object of the present invention is to provide an isolated, purified genomic DNA fragment comprising the nucleotide sequence shown in SEQ ID NO:1; plasmids and microorganisms containing said DNA fragment; a method of conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing the cDNA corresponding to the mRNA encoded by said DNA fragment that confers porphyric herbicide resistance on plant or algal cells, wherein said cDNA is expressed; and plants or algae into which cDNA corresponding to the mRNA encoded by said DNA fragment having the nucleotide sequence shown in SEQ ID NO:1 has been introduced, wherein said cDNA is expressed.
Yet further objects of the present invention include the use of any of the DNA fragments or biologically functional equivalents thereof disclosed herein as a genetic marker (for herbicide resistance), to produce a recombinant plasmid or transformed microorganism, to produce a probe useful in identifying related DNA sequences that confer resistance to porphyrin-accumulating type herbicides in plant and algal cells, and to produce plants or algae resistant to porphyrin-accumulating type herbicides.
The DNA fragments and biologically functional equivalents thereof of the present invention are hereinafter referred to as the xe2x80x9csubject nucleic acid fragmentsxe2x80x9d or xe2x80x9csubject DNA fragmentsxe2x80x9d. Specific individual fragments will be designated by their restriction sites and molecular sizes (kb).
The present invention includes plasmids containing the above-mentioned DNA fragments or their biologically functional equivalents (hereinafter referred to as the xe2x80x9csubject plasmidsxe2x80x9d), microorganisms containing these DNA fragments or their equivalents (hereinafter referred to as the xe2x80x9csubject microorganismsxe2x80x9d), plants or algae containing these DNA fragments or their equivalents (hereinafter referred to as the xe2x80x9csubject plantsxe2x80x9d), and methods for conferring resistance to porphyrin-accumulating type herbicides upon plant and algal cells by using these DNA fragments or their equivalents.
With regard to the terminology used herein, the term xe2x80x9cDNA fragmentsxe2x80x9d refers not only to the subject DNA fragments, but also to degenerate isomers and genetically equivalent modified forms of these fragments. xe2x80x9cDegenerate isomersxe2x80x9d is taken here to mean isomers whose nucleotide base sequence is degenerately related to the original fragments; that is, all nucleic acid fragments including the corresponding mRNA or corresponding cDNA, that contain essentially the same genetic information as the original fragments. xe2x80x9cGenetically equivalent modified formsxe2x80x9d is taken here to mean DNA fragments that may have undergone base changes, additions, or deletions, but which essentially contain the same inherent genetic information as the original fragments.
Specific examples of the latter include DNA fragments whose nucleotide sequence shows high homology to the subject nucleic acid fragments, which are readily isolated using conventional DNA-DNA or DNA-RNA hybridization techniques, or that are amplified using known PCR (Polymerase Chain Reaction) methods, and which possess the ability to confer resistance to porphyrin-accumulating type herbicides when introduced by conventional transformation techniques into plant or algal cells normally sensitive to these herbicides.
The phrase xe2x80x9cporphyrin-accumulating type herbicidexe2x80x9d or the phrase xe2x80x9cporphyric herbicidesxe2x80x9d refers to light-dependent herbicides, i.e., compounds that kill sensitive plants in the presence of light, but which exhibit no herbicidal activity in darkness, and which induce the accumulation of high levels of porphyrins in plants to which they have been applied. These herbicides include, for example, oxadiazon, flupropacil, [N-(4-chloro-2-fluoro-5-propargyloxy)phenyl-3,4,5,6-tetrahydrophtalimide (referred to below as compound A), the diphenyl ether herbicides such as acifluorfen, lactofen, oxyfluorfen, as well as the following: pentyl[2-chloro-5-(cyclohex-1-ene-1,2-dicarboximido)-4-fluor-phenoxy]acetate, 7-fluoro-6-[(3,4,5,6,)-tetra-hydrophthalimido]-4-(2-propynyl)-1,4-benzoxazin-3 (2H)-one (referred to below as xe2x80x9ccompound Bxe2x80x9d). 6-[(3,4,5,6-tetrahydro)pthalimido]-4-(2-propynyl)-1,4-benzoxazin-3 (2H)-one, 2-[7-fluoro-3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzox-azin-6-yl]perhydroimidazo[1,5-a)pyridine-1,3-dione, 2-[(4-chloro-2-fluoro-5-propargyloxy)phenyl]perhydro-1-H-1,2,4-triazolo-[1,2-a]pyridazine-1,3-dione, 2-[7-fluoro-3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl] 5,6,7,8-1,2,4-triazolo [4,3-a)pyridine-3H-one, 2-[3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-1-methyl-6-trifluoromethyl-2,4 (1H, 3H)-pyrimidine dione, 2-[6-fluoro-2-oxo-3-(2-propynyl)-2,3-dihydrobenzthiazol-5-yl]-3,4,5,6-tetrahydrophthalimide, 1-amino-2-[3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-6-tri-fluoromethyl-2,4 (1H, 3H)-pyrimidinedione, as well as analogs of these compounds.
The subject nucleic acid fragments may be constructed by the artificial synthesis of their nucleotide sequences; however, they are more typically isolated from a mutant strain of the unicellular green alga Chlamydomonas reinhardtii, designated RS-3, that is resistant to porphyrin-accumulating type herbicides. Said mutant strain RS-3 is stored at the Chlamydomonas Genetics Center (address: DCMB Group, Department of Botany, Box 91000, Duke University, Durham, N.C., 27708-1000, USA) under the entry number CC-2674. Thus, the mutant strain RS-3 is publicly available for distribution. As will be described below, the microorganisms that host the plasmids containing the subject nucleic acid fragments are also on deposit under the terms of the Budapest Treaty, and are thus freely available as well. The plasmids hosted by these microorganisms can be readily extracted using conventional techniques and the subject fragments recovered by reference to the restriction maps shown in FIGS. 1(a)-1(d). It would be possible, for example, to introduce specific alterations into these fragments using PCR or other site-directed mutagenesis techniques, or to use the subject nucleic acid fragments or their corresponding cDNAs, PCR products, or oligonucleotides as probes to isolate other DNA fragments exhibiting high homology to the subject nucleic acid fragments, and thus to generate homologs as discussed above.
Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. It should be understood, however, that the following detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.